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Generation of Switch Hybrid DNA Between Ig Heavy Chain-µ and Downstream Switch Regions in B Lymphocytes¹

Jürgen R. Müller^*,, Thomas Giese^*, Diane L. Henry, J. Frederic Mushinski^* and Kenneth B. Marcu^2,

^* Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794

Ig heavy chain isotype switching in B lymphocytes is known to be preceded by transcription of a portion of the particular heavy chain gene segment that is targeted for recombination. Here, we describe an active role for these transcripts in the switch recombination process. Using an in vitro assay that exposes an artificial switch-µ (Sµ) minisubstrate to switch region transcripts in the presence of nuclear extracts from switching cells, we demonstrate that free 3' ends of the Sµ sequence are extended onto switch region transcripts by reverse transcription. The activity was induced in splenic B lymphocytes upon activation with LPS or CD40 ligand. This in vitro process is thought to be relevant to in vivo class switching for two reasons: 1) although only one-third of the Sµ minisubstrate actually contains Sµ sequence, all crossovers between switch regions occurred in the Sµ portion; and 2) treatment of B lymphocytes with IL-4, which enriches for switching to S1, increases the ratio of Sµ-S1 to Sµ-S3 hybrids by 16% after LPS treatment and by 37% after CD40 ligand activation, implicating this Sµ-primed reverse transcription of switch region transcripts as a novel mechanism of regulating the specificity of isotype switching. Further evidence for an active role of switch region transcripts was obtained by expressing S RNA in trans in the Bcl₁B₁ B lymphoma line. Endogenous Sµ-S switch circles were detected in Bcl₁B₁ cells expressing exogenous S RNA but not in mock-transfected cells.

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